Males and females differ biologically, though the implications of these differences in disease pathogenesis are not well characterized. Generally, females generate a more robust immune response to challenge than males which correlates with more efficient clearance of pathogens. In the case of influenza A virus, however, females have a greater risk of mortality associated with infection than males, which may be a result of exacerbated pro- inflammatory responses. In our model system, inoculation of male and female C57BL/6 mice with the mouse- adapted influenza A virus, A/Puerto Rico/8/34 (PR8) (H1N1), results in greater morbidity and mortality in females than males, although viral replication kinetics are similar between the sexes. We also have demonstrated that male and female mice have dimorphic production of several pro-inflammatory chemokines and cytokines following influenza A infection which may be mediated by sex steroid hormones. One chemokine in particular, MCP-1, is significantly elevated in females as early as 3 days post-inoculation. MCP- 1, an essential chemokine in the immune response to influenza A virus and is responsible for recruitment of monocytes, TNF-a/inducible nitric oxide producing DCs (tipDCs), neutrophils, and lymphocytes into the lungs following infection. My proposal aims to elucidate the molecular, immunological, and hormonal mechanisms mediating MCP-1-induced pathogenesis in female, but not male, mice during influenza A virus infection. Specific Aim 1 will test the hypothesis that sex steroid hormones mediate increased MCP-1 production in females, which causes an excessive influx of immune cells, particularly tipDCs, into the lungs during influenza infection. We will examine MCP-1 concentrations, infiltration of tipDCs, and viral replication in the lungs of gonadally intact and hormone manipulated male and female C57BL/6 mice following inoculation with PR8. We also will confirm the role of MCP-1 by manipulating MCP-1 signaling using pharmacological manipulantions. Production of MCP-1 and other pro-inflammatory cytokines/chemokines can be inhibited by peroxisome proliferator-activated receptor gamma (PPARg) signaling which antagonizes the activation of transcription factors (e.g. NF-kB). The goal of Specific Aim 2 is to test the hypothesis that dimorphic PPARg expression modulates the differential production of MCP-1 in males and females following influenza A infection. We will measure the production of PPARg and activity (e.g. NFkB phosphorylation) in the lungs of male and female mice following PR8 inoculation and determine the contribution of sex steroid hormones through hormone manipulation. We will validate the effects of PPARg on dimorphic production of MCP-1, viral replication, and subsequent influenza A pathogenesis using PPARg agonists and antagonists. The recent H1N1 influenza A pandemic reveals a female-biased mortality, especially during pregnancy. These data and our preliminary studies support the need for an in depth evaluation of the effects of sex steroid hormones on immune responses during influenza A infection. PUBLIC HEALTH RELEVANCE: Data from the 1918 H1N1 influenza pandemic, H5N1 avian influenza cases, and the current 2009 H1N1 pandemic reveal that responses to influenza A infection differ between males and females;the mechanism mediating the sex differences, however, have yet to be elucidated. Our data, using a mouse model of H1N1 influenza A, demonstrate elevated cytokines and chemokines may contribute to female-biased morbidity and mortality during infection. The studies in this proposal will establish the immunological and hormonal mechanisms as well as the functional significance of increased immunopathology in females compared with males during influenza virus infection.